Proteins and Peptides at Surfaces and Membranes
Biopolymers such as peptides and proteins play very important roles at surfaces and interfaces. Adsorbed proteins are important for applications including biosensing and stabilization of emulsions, and can lead to undesirable effects such as biological fouling of medical implants. It is important to understand how the conformation and interaction between proteins changes at surfaces, and to develop new techniques to control these interactions. A particularly important type of surface is that of a biological membrane, and understanding the interaction of proteins and peptides with biological membranes is important for the development of novel antimicrobials, the treatment and prevention of diseases, and the preservation of biological membranes under highly stressed conditions.
The Dutcher Lab is involved in many different aspects of proteins and peptides at surfaces and membranes:
Single molecule pulling of surface-active proteins on different surfaces
Binding of proteins to a surface alters their conformation compared to that in solution. They can spread out, or not, on an interface in response to various forces such as electrostatics, as well as hydrophilic and hydrophobic effects. We have used atomic force microscopy (AFM) imaging and single molecule force spectroscopy (SMFS) to study beta-lactoglobulin (b-LG) molecules localized at the interface between oil droplets and water. To immobilize the oil droplets, we have mechanically trapped them in the pores of a filtration membrane. For this sample geometry, we have used SMFS to pull on the b-LG molecules, revealing changes in their conformation and oligomerization in response to in situ changes in pH. We have compared the results obtained for the oil droplet surface with those that we obtained previously for SMFS measurements of b-LG molecules adsorbed onto hydrophilic mica surfaces. For example, at neutral pH, we observe large differences between the results obtained for the two surfaces in the pulling force required to fully extend the molecules, the spacing between sawtooth peaks in the force-distance curves, and the oligomerization of the molecules. We have also investigated the effect of the curvature of a surface on the conformation and oligomerization of adsorbed proteins by creating highly-curved, hydrophobic surfaces that are stable in buffer. By controlling the surface curvature on the nanoscale, we can shift the balance between enthalpic and entropic interactions and modify the interaction between adsorbed proteins.
Interactions of dehydrin proteins with model membranes
Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Using a variety of techniques such as FTIR, surface pressure isotherms, ellipsometry and atomic force microscopy, we have investigated the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We have found that TsDHN-1 and TsDHN-2 gain secondary structure upon association with large unilamellar vesicles (LUVs) that mimic the plant plasma and organellar membranes in vitro, that zinc induces further disorder-to-order transitions under such conditions, and that phosphorylation of these proteins facilitates actin assembly. Reducing the temperature also appeared to induce and/or stabilize ordered secondary structure. These structural characteristics of TsDHN-1 and TsDHN-2 highlight their functions in facilitating cold and drought tolerance in T. salsuginea, by both membrane and cytoskeletal stabilization. This work is done in collaboration with the group of George Harauz in the Department of Molecular and Cellular Biology, Guelph.
Single molecule imaging of peptides in lipid matrix
We have used molecular resolution scanning tunneling microscopy (STM) to obtain striking images of gramicidin, a model antibacterial peptide, inserted into a phospholipid matrix. The resolution of the images is superior to that obtained in previous attempts to image gramicidin in a lipid environment using atomic force microscopy (AFM). This breakthrough has allowed visualization of individual peptide molecules surrounded by lipid molecules. We have observed several important features: the peptide molecules do not aggregate, the peptide molecules adopt a single conformation corresponding to a specific ion channel form, and the lipid molecules adjacent to the peptide molecules are systematically longer than those in the lipid matrix. These results constitute a new approach to obtain structural characteristics of antibiotic peptides in lipid assemblies that is necessary for the understanding of their biological activity. This work is done in collaboration with the group of Jacek Lipkowski in the Department of Chemistry, Guelph.
Electric field driven changes in conformation and orientation of proteins and peptides in model membranes
We use surface-sensitive spectroscopic techniques, such as an infrared technique known as polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and a surface-sensitive adaption of circular dichroism (CD), to study changes in the conformation and orientation of both the lipid and peptide components of the membrane in response to changes in the electric field applied across the membrane. In particular, we have investigated the properties of gramicidin incorporated into a DMPC matrix supported at a Au(111) electrode surface. In the first study, the matrix consisted of stacks of 10 bilayers supported at the gold electrode, and the potential-induced changes in the peptide conformation and orientation were investigated using circular dichroism (CD). In a second study, we provided a description of the potential controlled changes in the structure of a single mixed DMPC/GD bilayer supported at the gold electrode surface using PM-IRRAS. This allowed us to provide new and unique information about the properties of the peptide containing membrane exposed to static electric fields that are comparable to the fields acting on a natural biological membrane.This work is done in collaboration with the group of Jacek Lipkowski in the Department of Chemistry, Guelph.